Exogenous genes mostly random integrated in the genome of transgenic animals which generated by somatic cell cloning and embryo transplantation. The copy number of exogenous gene is one of the important factors which affect the expression level of exogenous gene and genetic stability in transgenic animals. As the identification index of genetic stability of transgenic animals, it is necessary to detect the copy number of the exogenous gene after the survival of transgenic animals. In order to test whether there is relationship between the copies of exogenous red fluorescence protein from Discosoma sp. (DsRed) gene, and its expression level in the transgenic Arbas cashmere goat (Capra hircus) genomic, we used the absolute qRT-PCR method which was more efficient highly sensitive and accurate than the traditional method of Southern blot to identify the copy number of exogenous gene to test the copy number of 3 different fragments in exogenous DsRed gene in 6 transgenic cashmere goats. The results showed that the copy number of Cytomegalovirus (CMV) promoter ranged from 2.80±0.38 to 13.68±0.16, DsRed gene from 1.34±0.04 to 11.84±0.02, joint of CMV promoter and DsRed gene from 1.58±0.04 to 19.22±0.38. The expression of exogenous DsRed gene mRNA in 6 transgenic cashmere goats was detected by relative qRT-PCR method. The exogenous gene in 3 different fragments of copy number data respectively using SPSS software for correlation analysis, the correlation coefficient P value was, CMV promoter, P=0.763; DsRed gene, P=0.665; joint of CMV promoter and DsRed gene: P=0.803. The P values of the 3 groups were all greater than 0.05, The results showed that in our detection of 6 transgenic Arbas cashmere goats in exogenous DsRed gene expression and the copy number of the exogenous gene had no significant correlation at 0.05 level. In addition, the reliability of the qRT-PCR method in the study of the copy number of exogenous genes in large transgenic livestock was verified: With good reproducibility and controllable error, it can be the preferred method to detect the copy number of exogenous gene in transgenic animals.

Abstract Elongase of very long chain fatty acids 6 (ELOVL6) is the rate-limiting enzyme in the elongation cycle in mammals that catalyzes the elongation of long-chain saturated and monounsaturated fatty acids such as 12–16 carbons. The objective of this study was to clone the active region of ELOVL6 promoter of Xinong Saanen Dairy Goat. Primers were designed based on genomic sequence of Yunnan black goat (Gene ID: NW_005100691.1 ), ELOVL6 promoter was cloned by PCR from goat genomic DNA. We used progressive deletion to get ten promoter fragments with different length , and then cloned into luciferase reporter gene vectors. The vectors and the pRL-TK were co-transfected into goat mammary epithelial cells. Using the dual-lueiferase reporter assay system to detect the expression activity．We obtained 2 370 bp（containing 2 168 bp upstream of the transcription start site）of 5' flanking sequence of ELOVL6 with 94%、81% and 80% homology with bovine, homo and mouse, respectively. Bioinformatics analysis showed that there was no typical transcription element TATA-box in ELOVL6 promoter. The core region of ELOVL6 promoter located in 109 to 40 bp upstream of the transcription start site and the potential negative control element was analyzed by deletion. Moreover, some consensus sites for PPAR, LXRA, SREBP-1, Sp1and NF-Y were identified in ELOVL6 promoter. Those result would lay a foundation for the study of function mechanism of ELOVL6 promoter.

The plant β-1,3-glucanase belongs to one of the important pathogenesis-related proteins (PR-proteins), which could be accumulated when induced by exogenous signal molecule. The Arachis hypogaea promoter of β-1,3-glucanase (Ah-Glu-Pro) has been proved to be an inducible promoter, and there might contain some cis-regulatory elements which could respond to exogenous signal molecule. In order to clarify the promoter's function and further analyze and confirm the key cis-regulatory elements within the promoter, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was conducted with peanut genomic DNA as template, and a 970 bp fragment was obtained successfully, named by Ah-Glu-Pro. Online function prediction by PLACE and PlantCARE showed that Ah-Glu-Pro contained some typical cis-elements, such as TATA box and CAAT box, some cis-acting elements which could respond to pathogens and salicylic acid (SA), such as GRWAAW, GT1-motif, W-box, RAV1AAT, INRNTPSADB, AMMORESIVDCRNIA1 and BIHD1OS. Five forward primers Glu-F, Glu-P4, Glu-P3, Glu-P2 and Glu-P1 at the 5' end with the XbaⅠrestriction enzyme cutting site and one reverse primer Glu-R at the 3' end with the BglⅡrestriction enzyme cutting site were designed and synthesized according to predictive results. PCR was conducted with Glu-F/ Glu-R, Glu-P4/Glu-R, Glu-P3/Glu-R, Glu-P2/Glu-R and Glu-P1/Glu-R, and Ah-Glu-P and 4 deleted fragments Ah-Glu-P4~Ah-Glu-P1 with the length of 931, 767, 650, 376 and 217 bp were obtained successfully, respectively. The 5 fragments were then transferred to plasmid pCAMBIA1301-xylA, replacing the Cauliflower mosaic virus 35S promoter (CaMV35S), and 5 corresponding plant expression vectors pCAMBIA1301-xylA-Glu-P~ pCAMBIA1301-xylA-Glu-P1 with xylose isomerase gene (xylA) as safe maker and with β-glucuronidase gene (GUS) as reporter gene were constructed successfully. These plant expression vectors were then transformed to Agrobacterium tumefaciens EHA105 using freezing and thawing method, and further transformed to onion (Allium cepa) epidermal cells by Agrobacterium mediated transformation. GUS histochemical staining and GUS enzyme activity in these transgenic onions were detected. GUS staining on the onion epidermal cells transformed by Ah-Glu-Pro showed that the cells appeared blue when were induced by 5.0 mmol/L SA, while appeared light blue without induction by SA. Those onion epidermal cells transformed by pCAMBIA1301-xylA-Glu-P4~pCAMBIA1301-xylA-Glu-P1 appeared blue when induced by 5.0 mmol/L SA. Those onion epidermal cells transformed by Ah-Glu-P~Ah-Glu-P4 appeared much deep blue than those cells transformed by Ah-Glu-P3~Ah-Glu-P1. GUS enzyme activity in onions transformed by Ah-Glu-P~Ah-Glu-P3 were increased of 1.45, 2.16 and 1.27 times, respectively, after SA induction than that without SA induction. GUS enzyme activity in onions transformed by Ah-Glu-P2~Ah-Glu-P1 had no significant difference between with and without SA induction. Consideration of the predictive cis-regulatory elements showed that RAV1AAT, MYBCOREATCYCB1 and INRNTPSADB within Ah-Glu-P, Ah-Glu-P4 and Ah-Glu-P3 might be positive cis-elements, and GT1-motif and AMMORESIVDCRNIA1 between Ah-Glu-P and Ah-Glu-P4 might be negative cis-elements. The further function confirmation on these cis-regulatory elements will provide theoretical basis for regulating the peanut endogenous β-1,3-glucanase gene efficient expression, improving the disease resistance of peanut, and effective utilization of inducible promoter in the process of peanut genetic transformation.

Wheat stripe rust or yellow rust, caused by an biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), is one of the important epidemic disease of wheat (Triticum aestivum) in China. Wheat stripe rust in China has shown high levels of genetic diversity and toxicity mutation. Research has shown that sexual reproduction of Pst can result in pathogenic mutation and produce new races. As the start of the sexual stage of wheat stripe rust, teliospore is an essential stage for completing Pst sexual reproduction. To analyse the proteomic changes of wheat leave infected by Pst at telial stage and investigate how the host plant respond to the sporification of teliospore. Proteins were extracted from leaves infected with stripe rust CYR32 and control group using trichloroacetic acid (TCA)/phenol extraction, and then separated by using two-dimensional electeophoresis (2-DE). Using PDQuest software analysis, 22 up-regulated spots (relative expression more than 1.5 folds) were observed in Suwon11 leaves inoculated with stripe rust CYR32 at telial stage. The up-regulated protein spots were selected for Orbitrap analysis and database searching. All these proteins were successfully identified and were mainly involved in glucose metabolism, anti-stress and plant senescence related metabolic pathways. There were 13 proteins involved in glucose metabolism, among which triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were key enzymes in the process of glycolysis, and isocitrate dehydrogenase participates in the Krebs cycle. Five proteins were involved in polysaccharide degradation and related metabolic, with the rest participating in ATP synthesis. There were 6 anti-stress related proteins, mainly involved in the removal of harmful reactive groups and the synthesis of osmolytes. The rest 3 proteins participate in plant senescence related metabolic pathways, among which both ATP-dependent Clp protease proteolytic subunit and cysteine protease precursor participate in the degradation of macromolecular protein under stress condition. In addition, the S-adenosylmethionine synthase involved in the synthesis of ethylene which played an important role in plant senescence. Proteome-level results was further investigated and confirmed using qRT-PCR and found that 16 protein genes were up-regulated at the transcription level (relative expression more than 1.5 folds). These results showed that the sporulation of Pst teliospores influences the expression of wheat proteins involved in glucose metabolism, anti-stress and plant senescence related metabolic pathways, and it was assumed that the formation of teliospore may be associated with plant senescence. This study provides some theoretical basis for the further insight into the plant-pathogen interaction of wheat stripe rust at telial stage , which is significant for revealing the mechanism of teliospore formation and the comprehensive understanding of sexual stage of Pst.

Transforming growth factor β (TGF-β) superfamily plays critical roles in immune cell functional regulation, cell proliferation and differentiation, tissue wound repair. The half-smooth tongue sole (Cynoglossus semilaevis), with excellent properties such as fast growth, individual large, meat delicious and nutrient-rich, is an important economic marine fish species in the North of China. It has become a new kind of excellent breeding object in recent years. But with the enlargement of the farming scale, a variety of infectious diseases is becoming more and more frequently, especially the bacterial disease such as the Vibrio harveyi infection, fast onset and spread quickly. These diseases bring great economic losses to the aquaculture industry. Therefore, in order to carry out the half-smooth tongue sole immune genetic research and explore its immune response mechanism with Vibrio harveyi infection, the TGF-β1 and TGF-β3 genes were cloned from liver in the half -smooth tongue sole. Sequence analysis showed that TGF-β1 and TGF-β3 amino acid sequence consisted of multiple N-glycosylation sites and a TGF-β family signature. Analysis of phylogenetic tree indicated that TGF-β1 and TGF-β3 of the half-smooth tongue sole had a high homology with that of fish. The qRT-PCR analysis showed that TGF-β1 and TGF-β3 expressed in a diverse array of tissues including heart, kidney, intestine, muscle, skin, brain, gill, liver and spleen of healthy half-smooth tongue sole and there were significant differences. The highest levels of TGF-β1 were detected in skin, the higher levels were discovered in kidney and the lowest levels were found in muscle. TGF-β3 expressed the highest in skin, higher in brain and the lowest in muscle. After the Vibrio harveyi infection, the expression levels of TGF-β1 showed uptrend in liver, peaked at 48 h after infection, with 3.17 times of the control group, then fell to control levels. In the spleen, TGF-β1 and TGF-β3 showed similar expression trend , rising in the first stage and then decreasing. The expression of TGF-β1 and TGF-β3 reached maximum after infection 24 and 12 h, respectively, with 1.70 times and 1.61 times of the control group (P＜0.05). In the kidney, the expression trend of TGF-β1 and TGF-β3 were still similar, both peaked at 12 h after infection, respectively, with 1.23 times and 1.42 times of the control group (P＜0.05), then showed a downward trend. In the gill, TGF-β1 expression quantity did not change significantly comparing with the control group. TGF-β3 expression quantity rose in the first stage and then decreased, peaked at 24 h after infection, with 4.71 times of the control group (P＜0.05). The above results suggested that TGF-β1 and TGF-β3 may play an important role in the immune against bacterial infections in the half-smooth tongue sole. This study provides a strong evidence for the participation of TGF-β1 and TGF-β3 in the immune regulation mechanism and also a theoretical basis for molecular immune research in the half- smooth tongue sole.

Barley stripe disease is caused by Pyrenophora graminea, and is a worldwide disease. To explore the interaction mechanism between Hordeum vulgare and Pyrenophora graminea, total protein of Ganpi 6 and Isotta leaves which were inoculated with Pyrenophora graminea (DWC) for 7 and 21 days were extracted to analyze proteomic changes. Twenty eight protein spots were found which showed a change of more than 1.4-fold between the inoculated and the mock-inoculated samples of Ganpi 6 and Isotta, of which 4 were up-regulated, 6 were down-regulated, 2 were induced and 2 were inhibited for Ganpi 6, and 3 were up-regulated, 4 were down-regulated, 4 were induced and 3 were inhibited for Isotta. The up-regulated proteins included ribulose-bisphosphate carboxylase activase A (No. 2 spot), actin (No. 9 spot), ribosome-recycling factor (RRF)(No. 10 spot), ATP synthase gamma chain(No. 11 and 27 spots), succinate dehydrogenase [ubiquinone] flavoprotein subunit (No. 15 spot) and predicted protein (No. 26 spot). The down-regulate protein spots included bas1 protein (No. 4 spot), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCo)(No. 3, 5, 12, 14, 16 and 18 spots), ATP synthase CF1 alpha subunit (No. 13 spot), Ycf3 (No. 17 spot) and alpha-1,4-glucan-protein synthase (UTPG)(No. 28 spot). The induced expression protein spots included predicted protein (No. 6 spot), lipoxygenase 2 (LOX2)(No. 7 spot), light harvesting chlorophyll a/b-binding protein (LHCP)(No. 19 spot), 3-phosphoglycerate kinase (PGK)(No. 20 spot), lectin (No. 21 spot) and ATP synthase gamma chain (No. 22 spot). The inhibited protein spots included RuBisCo (No. 1, 8, 24, 25 spots) and ribulose bisphosphate carboxylase small chain (RuBPCase)(No. 23 spot). These protein spots were identified by MALDI -TOF/ TOF MS and protein database searching by MASCOT program. The functions of the 28 protein were related to photosynthesis, protein synthesis, plant defense responses, cell signaling transduction, cellulose biosynthesis, and response to resist disease. These differentially expressed proteins might be related to the response to infection by Pyrenophora graminea. The results elucidate the proteomic basis of barley varieties with different resistance to Pyrenophora graminea.

Chinese cabbage (Brassica rapa subsp. pekinensis) will be subjected to mechanical damage during growing, cultivation, processing and transportation. In this study, Chinese cabbage was processed by injury treatment, then the production of jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) were detected using gas chromatography-mass spectrometry (GC-MS), GC and high performance liquid chromatography (HPLC), respectively. The transcripts of genes involved in wounding response were detected by cDNA microarray analysis. The expression level of the marker genes were measured by cDNA microarray analysis and semiquantitative RT-PCR. The results of cDNA microarray analysis showed that a large number of genes were up-regulated after wounding, which included transcription factor, small GTP-binding protein, receptor-like protein kinase, protein kinase, the genes for JA/ET biosynthesis, defence-related genes, the genes involved in glucosinolates degradation, the genes for tryptophan biosynthesis, the genes for modifying cell wall, the genes invovled in alkaloid and phenylpropanoid metabolic pathways and several molecular chaperones. The analysis of JA content showed that the production of JA was significantly higher in the wounding leaves than that in the non-wounding leaves at 6, 12, 24 and 48 h. Gene expression analysis showed that the key genes for JA biosynthesis which included allene oxide synthase (AOS), allene oxide cyclase (AOC), lipoxygenase (LOX), the phospholipase D (PLD) which occur in the upstream of JA biosynthesis and the JA-responsive marker gene, myrosinase-associated protein (MYAP) were up-regulated after wounding. The results indicated that JA signaling pathway might play an important role in the wounding response. The result of ET generation indicated that ET rapidly produced at 2 and 6 h after wounding treatment. Gene expression analysis for ET signal pathway showed that the key genes for ET biosynthesis, 1-aminocyclopropane-1- carboxylicacid (ACC) synthase 6 (ACS6) and ACC oxidase (ACO) were up-regulated at the early stage during wounding response process, and the ethylene-responsive transcription factor 4 (ERF4) was up-regulated at 0.17, 0.5, 2, 6, 12 and 24 h after wounding. These results indicated that ET signaling pathway participated in early wounding response. Both JA and ET required β-1,3 glucanase (BGL), and the expression of BGL was up-regulated at all time points which indicated that JA and ET coordinately regulated the wounding response. The contents of free and total SA were significantly decreased after wounding. The marker genes of SA signaling pathway, pathogenesis-related protein 1a (PR1a) and PR5, were down-regulated or unchanged at the early stages of wounding response, and up-regulated at the late stages during wounding response. It indicated that SA signaling pathway was suppressed at the early stage and activitied at the late stage, and involved in the defence response at the late stages of wounding response. This study clarified that some genes in the transcriptome were involved in the wounding response, and the production of JA, ET and SA and the expression level of the genes for JA, ET and SA signaling pathway were after wounding treatment. This finding is helpful to reveal the defense mechanism of the wounding response in Chinese cabbage.

Phosphoenolpyruvate carboxykinase 1 (Soluble) (PCK1) encodes the rate-limiting enzyme (phosphoenolpyruvate carboxykinase, cytosolic，PEPCK-C) in gluconeogenesis, and is the key gene at controlling the overall level of glucose. Several studies have reported that PCK1 had experienced selective adaption to environment changes. In this research, in order to study the expression differentiation of PCK1 during duck (Anas platyrhynchos domesticus) domestication, and analyze whether it had signature of selection adapted to the domestication, mallard (Anas platyrhynchos), Shaoxing ducks and Peking ducks were chosen as the representative of the wild breed, egg-type and meat-type ducks, respectively. Expression analysis found that PCK1 was down regulated in the 2 domesticated breeds. In comparison to the mallard, PCK1 was down-regulated 3- and 10- folds in the liver of Shaoxing ducks and Peking ducks, respectively and down-regulated 3- and 26- folds in the abdominal fat of Shaoxing ducks and Peking ducks, respectively. To identify whether PCK1 was selected during duck domestication, we performed whole-genome resequencing of mallards (n=27), Shaoxing ducks (n=27) and Peking ducks (n=27), and 12179415 SNP were obtained for genome wide analysis of selection signals, including analysis of the heterozygosity (Hp) and genetic distance (Fst). Z transformed whole-genome Hp (Z(Hp)) and Fst (Z(Fst)) distribution showed that Z(Hp)＜ -3 and Z(Fst)＞2 represented the extreme end of the distribution, and thus were described as the threshold of putatively selected signals. In this study, we only focus on selection signals in scaffold KB742479.1 which containing PCK1. And to reduce the interference of fixation due to genetic drift within populations, we combined results from Shaoxing ducks and Peking ducks and only chosen regions with reduced heterozygosity and increased genetic distance in both of the 2 breeds relative to Anas platyrhynchos. By using these thresholds KB742479.1:2 200 091~2 239 938 bp and KB742 479.1: 2 220 079~2 259 967 bp were identified. Comparing to KB742479.1:2 200 091~2 239 938 bp, the region of KB742479.1: 2 220 079~2 259 967 bp was found with lower Z(Hp) (-3.92 in Shaoxing ducks and -3.63 in Peking ducks) and higher Z(Fst) (3.91 in Shaoxing ducks and 2.54 in Peking ducks ), suggesting stronger selection signal was found in this region. KB742479.1: 2 220 079~2 259 967 bp was a genetic region, and further expression analysis of genes adjacent to this region, including an unknown downstream gene (Ensembl ID: ENSAPLG00000002901), upstream C20orf85 gene (chromosome 20 open reading frame 85), and PCK1, found that only PCK1 displayed differential expression during duck domestication. Then, to describe the extract distance between this region and PCK1, the full-length cDNA of PCK1 was cloned and blast against the duck genome. Results showed that PCK1 was mapped to the region of KB742479.1:2 342 320~2 350 143 bp which was 82.4 kb upstream from KB742 479.1: 2 220 079~2 259 967 bp. Based on these results we speculated that the selection signals happened at KB742479.1:2 342 320~2 350 143 bp might induce the differential expression of PCK1 during duck domestication. In summary, we presented the genetic mechanism of duck domestication for the first time, and the different expression of PCK1 and the selection signal 82.4 kb downstream from this gene obtained in this study will provide clues for the further function analysis of PCK1.

For mammals, ear is an important auditory organ for the recognition of sound waves and the identification of orientation. Considering that ear size of pig (Sus scrofa) has a very large variation after long-term domestication and artificial breeding, it is an ideal model animal for human auricular disease. So far, the study of pig ear size has mainly focused on the genetic mechanism, yet the research on the pig auricle cells is limited. In this study, using enzymatic method, cells in cartilage of pig ear was isolated and cultured in vitro. The cells in cartilage of pig ear were continuously subcultured and detected their size and distribution during the 3~10 passage by flow cytometry, respectively. The cells in cartilage of pig ear were mainly devided into fibrillar type (P1) and oval type (P2). P1 was of bigger size account for the vast majority of the cells (69.4%), and P2 was the minority (11.0%). Then the combination capacity of the 2 kinds of cells was detected using the positive markers of cartilage stem cells CD44 and CD90. The results showed that cells P1 could hardly combine with CD44, cells P2 had a strong combination capacity with CD44. However, both P1 and P2 could bind with CD90, which the binding capacity of P2 was slightly stronger than P1. According to the analysis results, pig auricle cells were comprised by chondrocytes (P1) and cartilage progenitor cells (P2). Subsequently, in order to study the quantity and physiological state changes of P1 and P2 cells during passages, the cells were incubated with FITC-Annexin V/PI to analyze apoptosis using flow cytometry. The result showed that apoptosis was a common phenomenon in the pig ear cartilage cells cutured in vitro from the 3~6 passages with a rising trend of apoptosis, and after the 6 generations, the situation of apoptosis was basically constant. Further apoptosis analysis result showed that P1 cells were only a small amount of apoptosis or necrosis, with cell viability of (96.61±1.32)%, indicating that P1 cell could maintain a stable state cultured in vitro. However, living cells accouts ＜16.5% in P2 cells from the 3~10 generation, and more than 83% of P2 cells were in the late stage of apoptosis or necrosis. This suggested that the P2 cells in the adult porcine ear were prone to apoptosis under in vitro culture conditions. As the number of passages increased and the time of culture prolonged, the proportion of P2 cells in primary cells decreased significantly. These limitations clearly illustrated the need to develop cell culture conditions which enabled prolonged expansion of cartilage progenitor cell. Meanwhile, the results also are valuable to the research on pig auricle as a model for human elastic cartilage regeneration medicine in the future.

Exploring genetic mechanism of chlorophyll biosynthesis and chloroplast development have important implication for high photosynthetic efficiency breeding in wheat (Triticum aestivum). In order to study the main physiological characterization, morphology and ultra-structure of chloroplast in a yellow-green leaf color mutant of common wheat, three near-isogenic lines (NILs) (a xantha line 1-20YY, a yellow-green line 1-20YG and a green line 1-20GG) which were derived from this yellow-green mutant were used by examination of the soluble substances content, a series of antioxidant enzymes activity, and morphology and ultra-structure of chloroplast. The results showed that the change of the leaf color had more influence on the content of soluble sugar and protein than that on the soluble proline. The content of soluble sugar and protein in the xantha line 1-20YY were significantly lower than in the green line 1-20GG and the yellow-green line 1-20YG except seedling stage (P＜0.05). And the content of soluble sugar and protein in the yellow-green line 1-20YG was no difference compared with the green line 1-20GG at all stages. Meanwhile, the yellowing of leaves also effected on activity of antioxidant enzymes. The influence on the activity of peroxidase (POD) was higher than that on the activity of superoxide dismutase (SOD) and hydrogen peroxidase (CAT). So this yellowing of leaves had serious influences to the content of soluble sugar and the activity of POD. From the chloroplast morphology, it could be found that the chloroplast shape of the xantha line 1-20YY was wad irregular and had a trend to the center of mesophyll cell. The chloroplast shape of the green line 1-20GG was nearly ellipse and the shape of chloroplast in 1-20YG line was almost spherical. The results of chloroplast ultra-structure with transmission electron microscopy revealed that the stroma thylakoids and grana thylakoids of the 1-20YY line were underdeveloped and their connection of each other were not tighter than that of 1-20YG line and 1-20GG line. There were no difference of the chloroplast ultra-structure between 1-20YG line and 1-20GG line. All these results provide a foundation for in-depth characterization of the formation mechanisms of the yellow-green leaf color mutant of wheat.

Genetically modified cotton (Gossypium hirsutum) event MON757 expressing Cry1Ac protein was developed by U.S. Monsanto Company targeting Lepidoptera pests. It has already been approved for planting or using for food/feed in the United States, Canada, Australia and other countries, but has not yet been permitted for planting or application in China. At present, there are not specific methods for Genetically Modified (GM) cotton MON757 reported to identify the homozygous/heterozygous states or to quantify its content in mixed samples. In order to detect GM cotton MON757 in commercial cotton seeds of China, we established the specific homozygous/heterozygous qualitative PCR method and the event-specific quantitative Real-time PCR (qRT-PCR) method for MON757 based on the DNA sequences of the junctions between exogenous DNA fragments and cotton genomic DNA. The homozygous/heterozygous qualitative PCR method using 3 primers respectively located in the exogenous DNA segments and the cotton genomic DNA sequence on both sides of the insertion fragments could accurately identify homozygous and heterozygous states of MON757. The qRT-PCR method established in this study could specifically detect the samples of MON757 with the limit of detection (LOD) about 11 copies and the limit of quantitative (LOQ) about 44 copies. We tested 49 samples of cotton seeds from the market in Hubei province of China, and 5 samples were identified having MON757. In order to quantify the MON757 content of those 5 samples, 600 seeds of each sample were mixed for quantitative test using event-specific qRT-PCR method. At the same time, we tested 60 seeds from each samples using the homozygous/heterozygous qualitative PCR method to calculate the MON757 content. The results of both methods showed that the content of MON757 of one sample was about 1.5% and other 4 samples are all over 20%. The homozygous/heterozygous PCR method and the qRT-PCR method for MON757 that we developed are suitable and useful for homozygous and heterozygous states identification of cotton plants and seeds in the field and for quantifying the content MON757 from mixed samples.

Litchi downy blight, caused by Peronophythora litchii, is one of the most destructive diseases affecting litchi (Litchi chinensis). Rapid and accurate detection of P. litchii is essential for early diagnosis and timely controlling the disease in the field. Based on the GTP-binding protein gene (Ypt1) of P. litchii, nested PCR and loop-mediated isothermal amplification (LAMP) primers were designed in this study. The reaction conditions were optimized, and the specificity and sensitivity were also assayed. For specificity testing, DNA extracted from 21 P. litchii isolates, 34 isolates of 13 closely related species of different oomycetes and 8 isolates of other fungal species were used in this study. The results showed that the PCR specific primer pair PvYF1/PvYR1 amplified an unique DNA fragment of 249 bp in all P. litchii isolates from geographically distinct counties in China, but there was no amplification product in any other non-P. litchii isolates. In addition, optimization of LAMP reaction conditions (temperature and time) revealed that the ideal settings for the primer set were 63 ℃ for 60 min, and a positive color (green) was only observed in the presence of P. litchii with calcein as a fluorescence indicator and the typical ladder-like banding pattern were observed on 2.0% gel electrophoresis in all DNA products of P. litchii. The identities of the amplified products of LAMP were also confirmed by direct sequencing, in which the sequences obtained were perfectly matched with the expected DNA sequences. The results also showed that the detection sensitivity was creased by 1000-fold to 100 fg/25 μL genomic DNA by developed a nested PCR, which amplified using Phytophthora genus Ypt1 universal primer pair Yph1F/Yph2R as the first round primers and PvYF1/PvYR1 as the second round primers. The detection sensitivity of LAMP assay was 10 fg/25 μL genomic DNA, which was up to 10 times higher than that of nested-PCR. Positive detection rates of P. litchii in diseased litchi samples collected from naturally field was also evaluated using conventional PCR, nested PCR and LAMP assays, and the isolations of P. litchii from these samples were verified using conventional isolation methods. Purified DNA was used as a positive control while sterile distilled water was used as a negative control. Finally, 30 infected and healthy looking but infested litchi leaves or fruits naturally and 3 healthy litchi leaves or fruits from Zhangzhou county were examined, the positive detection rate were 17/22 (77.3%) by conventional PCR, 20/22 (90.9%) by nested PCR, 21/22 (95.5%) by LAMP assay and 22/22 (100%) by conventional isolation method; 25 infected and healthy looking but infested litchi leaves or fruits naturally and 3 healthy litchi leaves or fruits from Putian county, the positive detection rate were 15/17 (88.2%) by conventional PCR, 16/17 (94.1%) by nested PCR, 17/17 (100%) by LAMP assay and 17/17 (100%) by conventional isolation method. Healthy litchi samples were inspected using conventional PCR, nested PCR, LAMP assay and traditional isolation methods, and all samples were negative for P. litchii. All reactions were repeated twice with consistent results. We conclused that LAMP is more sensitive, more specific and simpler than PCR, and the results can be visualized with the naked eye for the detection of P. litchii in resource-poor settings.